Project Summary Mycobacterium tuberculosis (Mtb) kills around 1.8 million people a year, more than any other infectious disease. The two main challenges of combating tuberculosis (TB) are the rapidly increasing number of multidrug-resistant clinical isolates, and the lack of drugs that completely sterilize Mtb infection. The latter is ascribed to the presence of persister cells that are not killed by antibiotics, and also evade the immune response. Mtb persisters are metabolically resting, non-replicating cells that reside in lung granulomas, compact aggregates of immune cells that are the hallmark of tuberculosis. The reduced vasculature of mature granulomas creates a microenvironment low in nutrients and oxygen that induces metabolic quiescence in Mtb. The exact mechanism of the transition to quiescence is unclear, but maintenance of redox homeostasis and oxidative phosphorylation via the electron transport chain appear to be essential to this process. The next generation of anti-TB chemotherapy should be a rational combination of highly active, synergistic drugs that kill both actively dividing cells and persister cells. The FDA-approval of the mycobacterial ATP synthase inhibitor bedaquiline has validated the energy generating machinery of Mtb as a viable drug target. Several new drug candidates (e.g. Q203) that inhibit the cytochrome (Cyt) bc1:aa3 complex, a component of the respiratory chain of Mtb, are in the pipeline. However, all bc1 inhibitors are bacteriostatic in Mtb. The scientific premise of this proposal is that the lack of cidal activity by this class of drugs is due to the presence of a second enzyme, the cytochrome bd oxidase (Cyt-bd). In addition to its role as a terminal oxygen reductase, Cyt-bd is required in cellular redox buffering in response to redox stressors. We hypothesize that (1) combined inhibition of Cyt-bd and Cyt-bc1:aa3 will abrogate terminal oxidation in Mtb, even in granulomas, and (2) inhibition of Cyt-bd will enhance efficacy of front-line and novel drugs to eradicate infection. Our preliminary results show that inactivation of Cyt-bd increases sensitivity to oxidative stress and to standard-of-care anti-TB drugs. We also demonstrated that Mtb lacking Cyt-bd is rapidly killed and cleared in mouse lungs treated with Q203 (Kalia et al. 2017, PNAS). Here, we propose to take the next step towards a new chemotherapeutic approach. In our first aim, we will investigate the synergistic lethality of terminal oxidase inhibition in an animal model that develops granulomatous lesions, similar to human tuberculosis. In aim two, we will evaluate synergies between anti-TB microbicides and inhibition of terminal oxidation. Finally, in aim three, we will focus on developing novel, small- molecule inhibitors of Cyt-bd that synergize with Q203, the pipeline Cyt-bc1:aa3 inhibitor. This will include structure-activity relationship studies, cytotoxicity and pharmacokinetics assessment and in vitro and in vivo potency testing. Successful completion of the proposed studies will contribute to combating TB drug resistance and to developing a sterilizing treatment against TB. !